title: Single-cell chemiluminescence imaging of parvovirus B19 life cycle.
abstract: Human parvovirus B19 (B19V) is a single-stranded DNA virus. The genome encodes a multifunctional non-structural protein (NS), two capsid proteins (VP1, VP2) and other small non-structural proteins (7.5 kDa, 9 kDa, 11 kDa). Within sensitive cells, B19V can achieve a productive replicative cycle or, on the contrary, establish persistence; differences in its expression profile have been yet investigated following in vitro infections by methodologies enabling information on the entire infected cell population. Conversely, the present study reports quantitative data on the production of B19V analytes (DNA, RNAs, and proteins) at single cell-level, underlining cell-to-cell differences through the viral specific macromolecular synthesis process. Microscope imaging assays (in situ hybridization and immunocytochemical assays), exploiting chemiluminescence as principle detection and targeting viral nucleic acids and antigens, have been performed on a permissive cell line following in vitro infection. Chemiluminescence, involving the emission of photons deriving from a chemical reaction, provided the localization and quantitative detection of analytes down to a few molecules within infected cells. In our experimental conditions, B19V transcriptional activity, leading to the production of NS and VP RNAs, has been detected early in the viral cycle (from 12h post-infection, hpi) and before genome replication, starting at 24 hpi. The analysis of VP RNAs and related proteins suggested an inhibitory effect on capsid protein translation, as a post-transcriptional regulation events. Indeed, high levels of VP transcripts have been detected at early stages of infection while the proteins accumulated within cells only at 48-72 hpi.
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy. Electronic address: francesca.bonvicini4@unibo.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bonvicini
authors.forename: Mara
authors.initials: M
authors.lastname: Mirasoli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Manaresi
authors.forename: Gloria
authors.initials: G
authors.lastname: Bua
authors.forename: Donato
authors.initials: D
authors.lastname: Calabria
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gallinella
mesh_terms: D036022:Capsid Proteins / Q000096:biosynthesis; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D049449:Luminescence; D008853:Microscopy; D061848:Optical Imaging / Q000379:methods*; D016732:Parvovirus B19, Human / Q000254:growth & development / Q000502:physiology*; D014176:Protein Biosynthesis; D059010:Single-Cell Analysis / Q000379:methods*; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins
keywords: B19V; Chemiluminescence; Immunocytochemical assay; In situ hybridization assay; Virus-specific macromolecular synthesis